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The Florida panther (Puma concolor coryi) is a relatively well studied species, but some aspects of its habitat requirements remain poorly understood. While it has been well established that the most important threat to panthers include limited habitat area and continued habitat loss and fragmentation, the importance of roads in this context has not been determined. The goal of this research is to determine the influence of roads on the movement patterns of the Florida panther. Panther telemetry data from 1981 until 2003 was used, as well as detailed road networks and vegetation maps. The influence of roads on individual panthers was determined through an analysis of: 1) vehicular mortality; 2) road crossing behavior; 3) road barrier effects; and 4) effectiveness of preventative measures. Results indicate that vehicle collisions continue to be a major threat to the Florida panther population, specifically adult males. Major roads form more of a barrier to movement than minor roads, but females are affected more than males. The combination of wildlife underpasses and high right-of-way fencing on I-75 has been extremely effective at vehicular mortality prevention, but the roadway remains a major barrier, particularly for female panthers. This has essentially segregated the movement of the sexes and has fragmented not only the limited habitat of the Florida panther, but also segments of the adult population critical to the propagation of the species.